Background In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7–8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR. Results Blastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05). Conclusions Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects.

Early embryonic development occurs in the oviduct, where an ideal microenvironment is provided by the epithelial cells and by the oviductal fluid produced by these cells. The oviductal fluid contains small extracellular vesicles (sEVs), which through their contents, including microRNAs (miRNAs), can ensure proper cell communication between the mother and the embryo. However, little is known about the modulation of miRNAs within oviductal epithelial cells (OECs) and sEVs from the oviductal fluid in pregnant cows. In this study, we evaluate the miRNAs profile in sEVs from the oviductal flushing (OF-sEVs) and OECs from pregnant cows compared to non-pregnant, at 120 h after ovulation induction. In OF-sEVs, eight miRNAs (bta-miR-126-5p, bta-miR-129, bta-miR-140, bta-miR-188, bta-miR-219, bta-miR-345-3p, bta-miR-4523, and bta-miR-760-3p) were up-regulated in pregnant and one miRNA (bta-miR-331-5p) was up-regulated in non-pregnant cows. In OECs, six miRNAs (bta-miR-133b, bta-miR-205, bta-miR-584, bta-miR-551a, bta-miR-1193, and bta-miR-1225-3p) were up-regulated in non-pregnant and none was up-regulated in pregnant cows. Our results suggest that embryonic maternal communication mediated by sEVs initiates in the oviduct, and the passage of gametes and the embryo presence modulate miRNAs contents of sEVs and OECs. Furthermore, we demonstrated the transcriptional levels modulation of selected genes in OECs in pregnant cows. Therefore, the embryonic-maternal crosstalk potentially begins during early embryonic development in the oviduct through the modulation of miRNAs in OECs and sEVs in pregnant cows.

Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell-to-cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle.Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 10 10 EVs/ml and 6.0 × 10 10 EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05).In UF-EVs, a total of 14 miRNAs were differentially abundant between stages.

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Background: In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by Size Exclusion Chromatography. Blastocyst rate was recorded on Days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism related transcripts and levels of active hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by pRT-PCR. Results: Blastocyst yield was lower (p<0.05) in BSA groups compared to dFCS groups. Survival rates after vitrification/warming were improved in dFCS-EVs compared to dFCS (p<0.05). EVs increased (p<0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared to dFCS and BSA groups, while lipid content was decreased and mitochondrial activity increased in dFCS-EV (p<0.05). Lipid metabolism transcripts were affected by EVs ( PPARGC1B and ACACA , p<0.05) and protein source in the medium ( CD36, PLIN2 and ATGL , p<0.05). Levels of pHSL were lower in dFCS (p<0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (p<0.05). Conclusions: Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, mitochondrial activity and relative changes in expression of lipid metabolism transcripts and lipase activation. Besides, EVs miRNA contents may contribute to the observed effects.